Isolating pond lives from vegetation – comparing 2 methods

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How do you isolate pond protists from the vegetation that they attach to, in an accurate/clean and efficient manner?

Many times I found most pond lives attached to vegetation - I had to put pieces of pond vegetation under microscope to obtain the most biodiversity.

When I took pond sample without vegetation, I often did not find much. I tried squeezing sample water out of pond vegetation and isolating bottom detritus, but still did not get many protists that way.

I found that some protists, such as vorticella, attach firmly to thick vegetation, making a clean isolation difficult.

To isolate pond lives from vegetation, I have compared two methods:

1) vegetation cut to fine pieces - Petri dish – pipette – cover slips

From my small tank (kept open-aired and under sun shine), I would remove/cut some pond vegetation and bottom detritus. Then I transfer them to a petri dish for observation under a compound scope with 2x-20x objective.

Vegetation has to be cut to very very small sizes, otherwise they would interfere with optical performance of objectives of 4x and up. No cover slips are used, because I would use a 200 micro liter micropipette to isolate interesting protists, if I see any in the petri dish. The isolated protest is then placed under cover slip for high magnification studies.

For 2x objective, condenser’s top lens can be removed to provide more even light cone, if desire.

For a regular 20x compound objective, the sample water layer should be thinner than 1 mm (a glass slide).

The drawback of this method is that protists would have to been seen/caught at 20x objective or lower. So some very small protists may be missed. Isolation is preferably done at 10x objective or lower, because a regular (non-LWD) 20x objective has working distance of less than 2mm. A LWD 20x objective is expensive (I have not seen a cheap one with LWD and good NA, please feel free to recommend any).

The advantage of this method is that I can isolate exactly what I want/see using a micropipette, and not worry about removing empty vegetation (those without protists attached). The suction of a 200 micro liter micropipette is accurate (between 20 – 200 micro liter), smooth and easily controlled (which is much easier to use than a pasteur pipette - even though I was trained as a biological researcher).

2) vegetation cut to fine pieces – 1.6 micron filter paper – rinse – cover slips


Basically I will filter cut vegetation with a 1.6 micron filter paper. Keep all filtrates and rinse them off with minimal amount of water. Then put that rinse water under cover slips for high magnification studies.

My 1.6 micron filter paper is this one:
http://www.amazon.com/Whatman-1820-042- ... n+1820-042

The drawback of this method is that empty vegetation/detritus (those without protisits attached) and sand will be retained by filter and may go under cover slips. And sometimes protists may attach to filter paper firmly and cannot not removed intact. Also filtering large amount of sample water with vegetation takes a long time. This method has no isolation accuracy to speak of – we basically have to isolate all particles.

The advantage of this method is not obvious, but since my filter’s pore size is 1.6 micron, I assume it will retain most protists.

I find myself using method 1) more and more.

I have read many past threads in the forum relevant to this topic, but did not find a method that is both highly accurate and efficient.

Your kind comments are highly appreciated.